Respiratory syncytial virus (RSV) is the leading cause of viral bronchiolitis and pneumonia worldwide, infecting more than 70% of children in the first year. RSV causes more frequent and severe infections in infants compared to adults. The development of vaccines has been complicated by the fact that host immune responses to RSV play a significant role in disease pathogenesis. A key to RSV pathogenesis may lie in defining the mechanisms associated with a deficient immune response at a time of immunological immaturity. The study of the responses of lung-resident myeloid cells to RSV in human infants has been limited due to the lack of available models. While it is possible to measure cytokines and chemokines present in tracheal aspirates in RSV bronchiolitis patients, it is difficult to obtain sufficient numbers of cells to perform mechanistic studies that might elucidate innate immune pathways that are defective in young children. Therefore, new models facilitating control of the timing of infection and cell manipulation are needed to compare the anti-RSV responses of neonatal and adult myeloid cells. An understanding of these mechanisms can aid in the design of new vaccines and therapies. Our long-term research goal is to use a 3D Human Tissue-Engineered Lung Model (3D-HTLM) that exhibits a normal immunological response against infectious agents to elucidate some of the viral and host determinants. The objective of this project is to create a 3D-HTLM that will be used to determine the mechanisms by which immunological immaturity leads to greater RSV pathogenesis. An advantage of the lung model is the ability to test the effect of immunological immaturity by comparing the response of young infants and children vs. adult immune cells to RSV infection. To achieve this goal, the 3D-HTLM will contain the cell types relevant to infection and an inflammatory response, including vascular endothelium, a respiratory epithelium, supporting stromal cells, and myeloid cells, both resident and inflammatory. The 3D-HTLM includes a 3D scaffold and extracellular matrix materials to allow for the correct cell physiological function and cell-to-cell interactions. We will pursue two specific aims. Aim 1: Determine if the lung microenvironment within the 3D-HTLM instructs the differentiation of lung resident myeloid cells. Aim 2: Compare the innate immune response of myeloid cells isolated from neonatal cord blood, young children and adults to RSV infection in the 3D-HTLM. The project will yield new information about the immune response that will provide new targets for preventative and therapeutic interventions of RSV infection, and the tissue-engineered lung model also may be used for testing RSV treatment strategies. In addition, expanding our knowledge about how cells interact with each other and with their environment will vertically advance the field of tissue engineering and the future development of an engineered lung for lung transplantation to treat a variety of lung diseases.